It is noted that protein transduction domains, which are called PTDs, may be used in researches on intracellular action mechanisms of proteins or efficient technology for applying proteins in clinical therapeutic agents, with in the early 1990s, knowing that some of proteins can enter into cells by penetrating cell membrane in living cells (Derossi et al., 1994, J. Biol. Chem., 269, 10444-10450). Particularly, such protein transduction domains may deliver materials such as nucleic acids or liposomes as well as macromolecules such as proteins into cells (Lewin et al., 2000, Nature Biotech., 18, 410-414; Torchilin, 2002, Cell. Mol. Biol. Lett., 7, 265-267).
Protein transduction domains known to date are peptides (TAT) derived from basic domains of HIV-1-derived TAT proteins, homeodomains (Antp) derived from Antennapedia of Drosophila, HSV-derived transcription factors (VP22) and synthetic lysine/arginine peptides, and the like (Wadia and Dowdy, 2003, Curr. Protein Pept. Sci., 4, 97-104). It was shown that among them, the most studied TAT peptide consisted of a domain of a total 11 basic amino acid domain from 47th to 57th amino acids (YGRKKRRQRRR, SEQ ID No. 7) in amino acid sequences of the TAT protein, and said TAT peptide only was also delivered alone or in the form of fused with other proteins into cell membranes (Vives et al., 1997, J. Biol. Chem., 272, 16010-16017). In addition, the TAT-PTD can deliver various proteins with a molecular weight in range of 10 kDa to 120 kDa, regardless of size, into cells (Fawell et al., 1994, Proc. Natl. Acad. Sci. USA., 91, 664-668; Nagahara et al., 1998, Nature Med., 4, 1449-1452). Moreover, it was identified that proteins fused with the TAT-PTD can be not only delivered into eukaryotic cells, but also have an inherent biological function of each protein in the cells and perform the function in the cells. The same result was reported even in an experiment using animal models such as a mouse (Asoh et al., 2002, Proc. Natl. Acad. Sci. USA., 99, 17107-17112; Cao et al., 2002, J. Neurosci., 22, 5423-5431).
To deliver proteins into cells by protein transduction domains is an independent mechanism, regardless of delivery methods previously known by receptors, transporters or endosomes, and it is assumed that their specific structural motifs or alignments by amino acids such as arginine or lysine affect on delivery ability of such protein transduction domains (Ho et al., 2001, Cancer Res., 61, 474-477). In addition, while knowing that only arginine oligomers have ability to be effectively delivered into cells, in contrast with histidine, lysine and the like, among positive charged amino acids, it is known that only electric charges of amino acids do not affect on intracellular delivery ability (Wender et al., 2000, Proc. Natl. Acad. Sci. USA., 97, 13003-13008). In case of intracellular delivery by HIV-1-derived TAT-PTD, the secondary structural motifs of amino acids such as α-helix play an important role, and it is reported that such α-helix structure maximizes substantially protein delivery efficiency (Ho et al., 2001, Cancer Res., 61, 474-477).
As described in the above, protein delivery peptides that deliver target proteins having intracellular functions into cells or tissues are efficient methods for delivering proteins, nucleic acids or drugs. Besides the first found TAT-PTD, various protein transduction domains had been found or synthesized. However, all conventionally known protein transduction domains are peptides derived or synthesized from virus and the like, and thus may substantially cause problems such as immune responses on applying them in human. Therefore, it is very important to find novel protein transduction domains derived from human that can replace the protein transduction domains known to date.
The inventors of the present invention found that some of full length amino acid sequences of human G protein α-12 served as transmembrane delivery peptides. Then, recombinant vectors coding said peptides were prepared, to which recombinant genes of target proteins were inserted so as to fuse with target proteins, followed by expressing and purifying proteins in Escherichia coli strains. Then, the proteins were delivered into animal cells. They identified this fact by biological methods to accomplish the present invention.